Gγ Subunits Research Articles

The role of Gαq in regulating NLRP3 inflammasome activation.

G proteins are a class of important signal transducers in mammalians. G proteins can corpoarated with G proteincoupled receptors (GPCRs) and transmit signals from extracellular stimuli into intracellular response, which will regulate a series of biological functions. G-proteins are heterotrimeric proteins composed of Gα, Gβ, and Gγ subunits. Based on structural and functional similarity of their α-subunits, G proteins are typically grouped into four classes (Gi, Gs, Gq/11, and G12/13). The Gq/11 subfamily consists of Gq, G11, G14, and G15/16 proteins. Gαq is the α-subunit of Gq protein and encoded by GNAQ. Our previous studies revealed that Gαq play an important role in regulating T cell survival and T cell differentiation. Inflammasomes are multiprotein complexes that play a critical role in modulating innate inflammatory response. NLRP3 inflammasome is currently the most extensively studied inflammasome. We found that Gαq suppressed NLRP3 inflammasome activation in macrophage, Gαq also suppressed NLRP3 inflammasome activation in a LPS-induced sepsis mouse model. Gαq can locate to mitochondria and Gαq was required for the maintenance of mitochondrial homeostasis. Gαq regulated NLRP3 inflammasome activation by modulating mitochondrial reactive oxygen species (mtROS). We found that Gαq suppressed NLRP3 inflammasome activation in macrophage, Gαq also suppressed NLRP3 inflammasome activation in a LPS-induced sepsis mouse model. Gαq can locate to mitochondria and Gαq was required for the maintenance of mitochondrial homeostasis. Gαq regulated NLRP3 inflammasome activation by modulating mitochondrial reactive oxygen species (mtROS). Our results indicate that Gαq regulates NLRP3 inflammasome activation by modulating mitochondrial ROS production. Our research provides new mechanistic insight into the activation of NLRP3 inflammasome. As it has been proved that NLRP3 inflammasome plays an important role in the pathogenesis many diseases such as Alzheimer's disease, cancer, and inflammatory bowel disease, Gαq might become a novel drug target for these diseases in future.

E3 ligase DECREASED GRAIN SIZE 1 promotes degradation of a G-protein subunit and positively regulates grain size in rice.

Grain size is one of the most important traits determining crop yield. However, the mechanism controlling grain size remains unclear. Here, we confirmed the E3 ligase activity of DECREASED GRAIN SIZE 1 (DGS1) in positive regulation of grain size in rice (Oryza sativa) suggested in a previous study. Rice G-protein subunit gamma 2 (RGG2), which negatively regulates grain size, was identified as an interacting protein of DGS1. Biochemical analysis suggested that DGS1 specifically interacts with canonical Gγ subunits (rice G-protein subunit gamma 1 [RGG1] and rice G-protein subunit gamma 2 [RGG2]) rather than non-canonical Gγ subunits (DENSE AND ERECT PANICLE 1 [DEP1], rice G-protein gamma subunit type C 2 [GCC2], GRAIN SIZE 3 [GS3]). We also identified the necessary domains for interaction between DGS1 and RGG2. As an E3 ligase, DGS1 ubiquitinated and degraded RGG2 via a proteasome pathway in several experiments. DGS1 also ubiquitinated RGG2 by its K140, K145 and S147 residues. Thus, this work identified a substrate of the E3 ligase DGS1 and elucidated the post transcriptional regulatory mechanism of the G-protein signalling pathway in the control of grain size.

GmAGB1 plays a positive regulatory role in soybean defense responses

Heterotrimeric GTP-binding protein (G-proteins) complex, which consists of Gα, Gβ and Gγ subunits, plays critical roles in defense signaling. Arabidopsis genome contains only a single Gβ-encoding gene, AGB1. Loss function of AGB1 in Arabidopsis results in enhanced susceptibility to a wide range of pathogens. However, the function of soybean AGB1 in immunity has not been previously interrogated. Bioinformatic analysis indicated that there are four GmAGB1 homologous genes in soybean genome, sharing homology of 86%-97%. To overcome the functional redundancy of these GmAGB1 homologs, virus-induced gene silencing (VIGS) mediated by the bean pod mottle virus (BPMV) was used to silence these four genes simultaneously. As expected, these four GmAGB1 homologous genes were indeed silenced by a single BPMV-VIGS vector carrying a conserved fragments among these four genes. A dwarfed phenotype was observed in GmAGB1s-silenced soybean plants, suggesting that GmAGB1s play a crucial role in growth and development. Disease resistance analysis indicated that silencing GmAGB1s significantly compromised the resistance of soybean plants against Xanthomonas campestris pv. glycinea (Xag). This reduced resistance was correlated with the decreased accumulation of pathogen-induced reactive oxygen species (ROS) and the reduced activation of GmMPK3 in response to flg22, a conserved N-terminal peptide of flagellin protein. These results indicate that GmAGB1 functions as a positive regulator in disease resistance and GmAGB1 is indispensable for the ROS production and GmMPK3 activation induced by pathogen infection. Yeast two hybrid assay showed that GmAGB1 interacted with GmAGG1, suggesting that an evolutionary conserved heterotrimeric G protein complex similarly functions in soybean.

Pea G-protein γ subunits: Unlocking their potential in physiological stress and mycorrhizal mediated nutrient sensing

Heterotrimeric GTP-binding proteins or G-proteins are pivotal players in the intricate signaling cascades of plant cells, operating through their binding to guanine nucleotides. These G-proteins primarily consist of three essential subunits: Gα, Gβ, and Gγ. Among these subunits, Gγ stands out for its remarkable genetic diversity. In the present investigation, six Gγ subunits were identified in the Indian variety T-163 of the pea plant (Pisum sativum). Notably, two of these novel Gγ subunits, named PsGγ1 and PsGγ2, belong to type A, while PsGγ3 falls within the type B category. The remaining three subunits, namely PsGγ4, PsGγ5, and PsGγ6, are classified under type C. An in-depth comparison of the amino acid sequences of these pea Gγ subunits with their counterparts in other plants, including Arabidopsis thaliana and Oryza sativa, has unveiled significant variations. This research explores the impact of different treatments on PsGγ genes (PsGγ1 to PsGγ6) in plants. Noteworthy discoveries include a 4-fold increase in the expression of PsGγ1, PsGγ4, PsGγ5, and PsGγ6 in the presence of nitrogen. PsGγ2 and PsGγ3, however, show no response. Phosphorus induces a 3-fold upregulation in PsGγ2 and PsGγ4, and a 4-fold increase in PsGγ5. Conversely, the absence of phosphorus triggers a 4-fold upregulation in PsGγ4 and PsGγ5. Heat stress leads to a 3-fold upregulation in PsGγ2, PsGγ4, and PsGγ5, while cold stress results in a 3-fold upregulation of PsGγ1 and PsGγ6. Under high salt conditions, PsGγ1, PsGγ3, PsGγ4, and PsGγ6 exhibit a 4-fold upregulation, with PsGγ2 showing a 2-fold increase. PsGγ4 and PsGγ5 display a 4-fold upregulation in response to ABA, while PsGγ2 and PsGγ3 show a 3-fold increase while MeJA induces a 4-fold upregulation in PsGγ5. Notably, this study unveils, for the first time, the significant role of Gγ subunits during endosymbiotic associations with phosphorus-acquiring AMF with AMF triggering a 4-fold upregulation in PsGγ4 and PsGγ6. The presence of multiple Gγ subunits in pea underscores their critical participation in governing plant development, stress responses, nutrient sensing, and interactions with mycorrhizal fungi.

Isolation and conformational analysis of the Gα α-helical domain

Heterotrimeric G proteins (G proteins), composed of Gα, Gβ, and Gγ subunits, are the major downstream signaling molecules of the G protein-coupled receptors. Upon activation, Gα undergoes conformational changes both in the Ras-like domain (RD) and the α-helical domain (AHD), leading to the dissociation of Gα from Gβγ and subsequent regulation of downstream effector proteins. Gα RD mediate the most of classical functions of Gα. However, the role of Gα AHD is relatively not well elucidated despite its much higher sequence differences between Gα subtypes than those between Gα RD. Here, we isolated AHD from Gαs, Gαi1, and Gαq to provide tools for examining Gα AHD. We investigated the conformational dynamics of the isolated Gα AHD compared to those of the GDP-bound Gα. The results showed higher local conformational dynamics of Gα AHD not only at the domain interfaces but also in regions further away from the domain interfaces. This finding is consistent with the conformation of Gα AHD in the receptor-bound nucleotide-free state. Therefore, the isolated Gα AHD could provide a platform for studying the functions of Gα AHD, such as identification of the Gα AHD-binding proteins.

An emerging role of heterotrimeric G-proteins in nodulation and nitrogen sensing.

A comprehensive understanding of nitrogen signaling cascades involving heterotrimeric G-proteins and their putative receptors can assist in the production of nitrogen-efficient plants. Plants are immobile in nature, so they must endure abiotic stresses including nutrient stress. Plant development and agricultural productivity are frequently constrained by the restricted availability of nitrogen in the soil. Non-legume plants acquire nitrogen from the soil through root membrane-bound transporters. In depleted soil nitrogen conditions, legumes are naturally conditioned to fix atmospheric nitrogen with the aid of nodulation elicited by nitrogen-fixing bacteria. Moreover, apart from the symbiotic nitrogen fixation process, nitrogen uptake from the soil can also be a significant secondary source to satisfy the nitrogen requirements of legumes. Heterotrimeric G-proteins function as molecular switches to help plant cells relay diverse stimuli emanating from external stress conditions. They are comprised of Gα, Gβ and Gγ subunits, which cooperate with several downstream effectors to regulate multiple plant signaling events. In the present review, we concentrate on signaling mechanisms that regulate plant nitrogen nutrition. Our review highlights the potential of heterotrimeric G-proteins, together with their putative receptors, to assist the legume root nodule symbiosis (RNS) cascade, particularly during calcium spiking and nodulation. Additionally, the functions of heterotrimeric G-proteins in nitrogen acquisition by plant roots as well as in improving nitrogen use efficiency (NUE) have also been discussed. Future research oriented towards heterotrimeric G-proteins through genome editing tools can be a game changer in the enhancement of the nitrogen fixation process. This will foster the precise manipulation and production of plants to ensure global food security in an era of climate change by enhancing crop productivity and minimizing reliance on external inputs.

Beyond the G protein α subunit: investigating the functional impact of other components of the Gαi3 heterotrimers

BackgroundSpecific interactions between G protein-coupled receptors (GPCRs) and G proteins play a key role in mediating signaling events. While there is little doubt regarding receptor preference for Gα subunits, the preferences for specific Gβ and Gγ subunits and the effects of different Gβγ dimer compositions on GPCR signaling are poorly understood. In this study, we aimed to investigate the subcellular localization and functional response of Gαi3-based heterotrimers with different combinations of Gβ and Gγ subunits.MethodsLive-cell imaging microscopy and colocalization analysis were used to investigate the subcellular localization of Gαi3 in combination with Gβ1 or Gβ2 heterotrimers, along with representative Gγ subunits. Furthermore, fluorescence lifetime imaging microscopy (FLIM-FRET) was used to investigate the nanoscale distribution of Gαi3-based heterotrimers in the plasma membrane, specifically with the dopamine D2 receptor (D2R). In addition, the functional response of the system was assessed by monitoring intracellular cAMP levels and conducting bioinformatics analysis to further characterize the heterotrimer complexes.ResultsOur results show that Gαi3 heterotrimers mainly localize to the plasma membrane, although the degree of colocalization is influenced by the accompanying Gβ and Gγ subunits. Heterotrimers containing Gβ2 showed slightly lower membrane localization compared to those containing Gβ1, but certain combinations, such as Gαi3β2γ8 and Gαi3β2γ10, deviated from this trend. Examination of the spatial arrangement of Gαi3 in relation to D2R and of changes in intracellular cAMP level showed that the strongest functional response is observed for those trimers for which the distance between the receptor and the Gα subunit is smallest, i.e. complexes containing Gβ1 and Gγ8 or Gγ10 subunit. Deprivation of Gαi3 lipid modifications resulted in a significant decrease in the amount of protein present in the cell membrane, but did not always affect intracellular cAMP levels.ConclusionOur studies show that the composition of G protein heterotrimers has a significant impact on the strength and specificity of GPCR-mediated signaling. Different heterotrimers may exhibit different conformations, which further affects the interactions of heterotrimers and GPCRs, as well as their interactions with membrane lipids. This study contributes to the understanding of the complex signaling mechanisms underlying GPCR-G-protein interactions and highlights the importance of the diversity of Gβ and Gγ subunits in G-protein signaling pathways.C8a11hmRuxdWs3DubYRKG2Video

An atypical heterotrimeric Gα and its interactome suggest an extra-large role in overcoming abiotic and biotic stress.

Canonical heterotrimeric G-proteins (G-proteins) are comprised of Gα, Gβ, and Gγ subunits. G-proteins regulate multiple crucial plant growth and development processes, incorporating environmental responses. Besides Gα, Gβ and Gγ, the discovery of atypical Gα subunits termed as extra-large G-proteins or extra-large GTP-binding proteins (XLGs) makes G-protein signaling unique in plants. The C-terminus of XLG shares similarities with the canonical Gα subunits; the N-terminus harbors a nuclear localization signal (NLS) and is rich in cysteine. The earlier explorations suggest XLG's role in flowering, the development of embryos and seedlings, root morphogenesis, stamen development, cytokinin-induced development, stomatal opening and regulation of rice grain filling. The XLGs are also known to initiate signaling cascades that prime plants against a variety of abiotic and biotic stresses. They are also engaged in controlling several agronomic parameters such as rice panicle length, grain filling, grain size, and biomass, highlighting their potential contribution to crop improvement. The present review explores the remarkable properties of non-canonical Gα subunits (XLGs) and reflects on the various developmental, abiotic and biotic stress signaling pathways controlled by them. Moreover, the bottleneck dilemma of how a tiny handful of XLGs control a multiplicity of stress-responsive activities is partially resolved in this review by addressing the interaction of XLGs with different interacting proteins. XLG proteins presented in this review can be exploited to gain access to highly productive and stress-tolerant plants.

CaaX-motif-adjacent residues influence G protein gamma (Gγ) prenylation under suboptimal conditions

Prenylation is an irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Dysregulation of prenylation contributes to multiple disorders, including cancers and vascular and neurodegenerative diseases. Prenyltransferases tether isoprenoid lipids to proteins via a thioether linkage during prenylation. Pharmacological inhibition of the lipid synthesis pathway by statins is a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential inhibition. We examined the prenylation of carboxy-terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to prenylation and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide, statin sensitivity, and extent of prenylation. Our results also show a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings indicate a plausible mechanism allowing for statins to differentially perturb heterotrimeric G protein signaling in cells depending on their Gγ-subtype composition. Our results may also provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.

Extra-large G proteins have extra-large effects on agronomic traits and stress tolerance in maize and rice.

Heterotrimeric G proteins - comprising Gα, Gβ, and Gγ subunits - are ubiquitous elements in eukaryotic cell signaling. Plant genomes contain both canonical Gα subunit genes and a family of plant-specific extra-large G protein genes (XLGs) that encode proteins consisting of a domain with Gα-like features downstream of a long N-terminal domain. In this review we summarize phenotypes modulated by the canonical Gα and XLG proteins of arabidopsis and highlight recent studies in maize and rice that reveal dramatic phenotypic consequences of XLG clustered regularly interspaced short palindromic repeats (CRISPR) mutagenesis in these important crop species. XLGs have both redundant and specific roles in the control of agronomically relevant plant architecture and resistance to both abiotic and biotic stresses. We also point out areas of current controversy, suggest future research directions, and propose a revised, phylogenetically-based nomenclature for XLG protein genes.

GR5 acts in the G protein pathway to regulate grain size in rice

Grain size is an important determinant of grain yield in rice. Although dozens of grain size genes have been reported, the molecular mechanisms that control grain size remain to be fully clarified. Here, we report the cloning and characterization of GR5 (GRAIN ROUND 5), which is allelic to SMOS1/SHB/RLA1/NGR5 and encodes an AP2 transcription factor. GR5 acts as a transcriptional activator and determines grain size by influencing cell proliferation and expansion. We demonstrated that GR5 physically interacts with five Gγ subunit proteins (RGG1, RGG2, DEP1, GS3, and GGC2) and acts downstream of the G protein complex. Four downstream target genes of GR5 in grain development (DEP2, DEP3, DRW1, and CyCD5;2) were revealed and their core T/CGCAC motif identified by yeast one-hybrid, EMSA, and ChIP–PCR experiments. Our results revealed that GR5 interacts with Gγ subunits and cooperatively determines grain size by regulating the expression of downstream target genes. These findings provide new insight into the genetic regulatory network of the G protein signaling pathway in the control of grain size and provide a potential target for high-yield rice breeding.

N-terminal intrinsic disorder is an ancestral feature of Gγ subunits that influences the balance between different Gβγ signaling axes in yeast

Activated G protein-coupled receptors promote the dissociation of heterotrimeric G proteins into Gα and Gβγ subunits that bind to effector proteins to drive intracellular signaling responses. In yeast, Gβγ subunits coordinate the simultaneous activation of multiple signaling axes in response to mating pheromones, including MAP kinase (MAPK)-dependent transcription, cell polarization, and cell cycle arrest responses. The Gγ subunit in this complex contains an N-terminal intrinsically disordered region that governs Gβγ-dependent signal transduction in yeast and mammals. Here, we demonstrate that N-terminal intrinsic disorder is likely an ancestral feature that has been conserved across different Gγ subtypes and organisms. To understand the functional contribution of structural disorder in this region, we introduced precise point mutations that produce a stepwise disorder-to-order transition in the N-terminal tail of the canonical yeast Gγ subunit, Ste18. Mutant tail structures were confirmed using circular dichroism and molecular dynamics and then substituted for the wildtype gene in yeast. We find that increasing the number of helix-stabilizing mutations, but not isometric mutation controls, has a negative and proteasome-independent effect on Ste18 protein levels as well as a differential effect on pheromone-induced levels of active MAPK/Fus3, but not MAPK/Kss1. When expressed at wildtype levels, we further show that mutants with an alpha-helical N terminus exhibit a counterintuitive shift in Gβγ signaling that reduces active MAPK/Fus3 levels whilst increasing cell polarization and cell cycle arrest. These data reveal a role for Gγ subunit intrinsically disordered regions in governing the balance between multiple Gβγ signaling axes.

Yeast Chaperone Hsp70-Ssb Modulates a Variety of Protein-Based Heritable Elements.

Prions are transmissible self-perpetuating protein isoforms associated with diseases and heritable traits. Yeast prions and non-transmissible protein aggregates (mnemons) are frequently based on cross-β ordered fibrous aggregates (amyloids). The formation and propagation of yeast prions are controlled by chaperone machinery. Ribosome-associated chaperone Hsp70-Ssb is known (and confirmed here) to modulate formation and propagation of the prion form of the Sup35 protein [PSI+]. Our new data show that both formation and mitotic transmission of the stress-inducible prion form of the Lsb2 protein ([LSB+]) are also significantly increased in the absence of Ssb. Notably, heat stress leads to a massive accumulation of [LSB+] cells in the absence of Ssb, implicating Ssb as a major downregulator of the [LSB+]-dependent memory of stress. Moreover, the aggregated form of Gγ subunit Ste18, [STE+], behaving as a non-heritable mnemon in the wild-type strain, is generated more efficiently and becomes heritable in the absence of Ssb. Lack of Ssb also facilitates mitotic transmission, while lack of the Ssb cochaperone Hsp40-Zuo1 facilitates both spontaneous formation and mitotic transmission of the Ure2 prion, [URE3]. These results demonstrate that Ssb is a general modulator of cytosolic amyloid aggregation, whose effect is not restricted only to [PSI+].

Gγ subunit AT1/GS3-the “code” of alkaline tolerance in main graminaceous crops

This brief article highlights the results of Zhang et al. (Science 379, eade8416, 2023), who recently found that the Gγ subunit AT1/GS3 contributes to alkaline tolerance in several main monocots crops, and revealed the molecular mechanism of AT1/GS3-mediated response to alkaline stress in plants, which involves regulating H2O2 levels by inhibiting the phosphorylation of aquaporin PIP2s.

Genetic modification of Gγ subunit AT1 enhances salt-alkali tolerance in main graminaceous crops.

Genetic modification of Gγ subunit AT1 enhances salt-alkali tolerance in main graminaceous crops.

Gβγ signaling regulates microtubule-dependent control of Golgi integrity

Gβγ subunits regulate several non-canonical functions at distinct intracellular organelles. Previous studies have shown that Gβγ signaling at the Golgi is necessary to mediate vesicular protein transport function and to regulate mitotic Golgi fragmentation. Disruption of Golgi structure also occurs in response to microtubule depolymerizing agents, such as nocodazole. In this study, we use siRNA against Gβ1/2 or specific Gγ subunits to deplete their expression, and show that their knockdown causes a significant reduction in nocodazole-induced Golgi fragmentation. We establish that knockdown of Gβγ or inhibition of Gβγ with gallein resulted in decreased activation of protein kinase D (PKD) in response to nocodazole treatment. We demonstrate that restricting the amount of free Gβγ available for signaling by either inhibiting Gαi activation using pertussis toxin or by knockdown of the non-GPCR GEF, Girdin/GIV protein, results in a substantial decrease in nocodazole-induced Golgi fragmentation and PKD phosphorylation. Our results also indicate that depletion of Gβγ or inhibition with gallein or pertussis toxin significantly reduces the microtubule disruption-dependent Golgi fragmentation phenotype observed in cells transfected with mutant SOD1, a major causative protein in familial amyotrophic lateral sclerosis (ALS). These results provide compelling evidence that Gβγ signaling is critical for the regulation of Golgi integrity.

Extra-large G proteins regulate disease resistance by directly coupling to immune receptors in Nicotiana benthamiana

Heterotrimeric G proteins, comprising Gα, Gβ, and Gγ subunits, are key regulators of eukaryotic intracellular signaling. Extra-large G (XLG) proteins are a subfamily of plant-specific Gα proteins interacting with plasma membrane-localized receptors to regulate multiple biological processes. The Nicotiana benthamiana genome encodes seven XLG proteins, NbXLG1–7, whose functions in disease resistance and underlying mechanisms are unknown. In this study, we silenced all the seven genes and found that disease susceptibility was enhanced when both NbXLG3 and NbXLG5 or NbXLG4 was silenced. Then, we generated N. benthamiana xlg3xlg5 double- and xlg4 single-mutant lines using the CRISPR-Cas9 approach. All the mutants showed reduced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, the fungal pathogen Sclerotinia sclerotiorum, and a series of oomycete pathogens, including Phytophthora capsici, Phytophthora infestans, and Phytophthora parasitica. We further demonstrated that NbXLG3/4/5 positively regulated microbial pattern-induced reactive oxygen species burst and defense gene expression by directly coupling to the tested plant immune receptors. In addition, we examined the role of NbXLG3/4/5 in abiotic stress tolerance and observed that NbXLG3 and NbXLG5 negatively regulated plant resistance to high-salt, mannitol, and PEG. Our study demonstrates the possible role of NbXLG3/4/5 in response to biotic and abiotic stresses and provides insights for the improvement of plant resistance to environmental changes.

Regulation of rod photoreceptor function by farnesylated G-protein γ-subunits.

Heterotrimeric G-protein transducin, Gt, is a key signal transducer and amplifier in retinal rod and cone photoreceptor cells. Despite similar subunit composition, close amino acid identity, and identical posttranslational farnesylation of their Gγ subunits, rods and cones rely on unique Gγ1 (Gngt1) and Gγc (Gngt2) isoforms, respectively. The only other farnesylated G-protein γ-subunit, Gγ11 (Gng11), is expressed in multiple tissues but not retina. To determine whether Gγ1 regulates uniquely rod phototransduction, we generated transgenic rods expressing Gγ1, Gγc, or Gγ11 in Gγ1-deficient mice and analyzed their properties. Immunohistochemistry and Western blotting demonstrated the robust expression of each transgenic Gγ in rod cells and restoration of Gαt1 expression, which is greatly reduced in Gγ1-deficient rods. Electroretinography showed restoration of visual function in all three transgenic Gγ1-deficient lines. Recordings from individual transgenic rods showed that photosensitivity impaired in Gγ1-deficient rods was also fully restored. In all dark-adapted transgenic lines, Gαt1 was targeted to the outer segments, reversing its diffuse localization found in Gγ1-deficient rods. Bright illumination triggered Gαt1 translocation from the rod outer to inner segments in all three transgenic strains. However, Gαt1 translocation in Gγ11 transgenic mice occurred at significantly dimmer background light. Consistent with this, transretinal ERG recordings revealed gradual response recovery in moderate background illumination in Gγ11 transgenic mice but not in Gγ1 controls. Thus, while farnesylated Gγ subunits are functionally active and largely interchangeable in supporting rod phototransduction, replacement of retina-specific Gγ isoforms by the ubiquitous Gγ11 affects the ability of rods to adapt to background light.

Molecular Identification of the G-Protein Genes and Their Expression Profiles in Response to Nitrogen Deprivation in Brassica napus.

Heterotrimeric guanine nucleotide binding protein (G-protein) consisting of Gα, Gβ, and Gγ subunits is one of the key signal transducers in plants. Recent studies indicated that G-protein has been proposed as an important mediator of nitrogen responses in rice, wheat, and Arabidopsis. However, little is known about these G-proteins in Brassica napus (B. napus), except for three identified G-proteins, BnGA1, BnGB1, and BnGG2. Therefore, the aim of the present study is to characterize the members of the G-protein gene family in allotetraploid B. napus and to analyze their expression profiles in response to nitrogen deprivation. In total, 21 G-protein family members were identified in B. napus, encoding two Gα, six Gβ, and 13 Gγ. Sequence and phylogenetic analyses showed that although genome-wide triploid events increased the number of genes encoding Gα, Gβ, and Gγ subunits, the gene structure and protein properties of the genes encoding each G-protein subunit were extremely conserved. Collinearity analysis showed that most G-protein genes in B. napus had syntenic relationships with G-protein members of Arabidopsis, Brassica rape (B. rapa), and Brassica oleracea (B. oleracea). Expression profile analysis indicated that Gα and C-type Gγ genes (except BnGG10 and BnGG12 were highly expressed in flower and ovule) were barely expressed in most organs, whereas most Gβ and A-type Gγ genes tended to be highly expressed in most organs. G-protein genes also showed various expression patterns in response to nitrogen-deficient conditions. Under nitrogen deficiency, Gα and five C-type Gγ genes were upregulated initially in roots, while in leaves, Gα was downregulated initially and five C-type Gγ genes were highly expressed in different times. These results provide a complex genetic dissection of G-protein genes in B. napus, and insight into the biological functions of G-protein genes in response to nitrogen deficiency.

G-protein couples MAPK cascade through maize heterotrimeric Gβ subunit.

G protein couples MAPK cascade through maize heterotrimeric Gβ subunit MGB1. Heterotrimeric G protein Gβ interacts with Gγ subunit to generate Gβγ dimer in modulation of various biological processes. The modulatory events at transcriptome scale of plant Gβ subunit remain largely unknown. To reveal the regulatory basis of Gβ subunit at transcriptome level, we first identified a canonical maize Gβ subunit MGB1 that physically interacted with Type C Gγ protein MGG4. For transcriptome analysis, two independent CRISPR/Cas9-edited MGB1 lines were generated, which all exhibited growth arrest, suggestive of MGB1 essential for maize seedling establishment. Transcriptomic outcomes showed that MGB1 knockout resulted in elevated transcriptional abundance of plant immune response marker PR and immune receptor RPM1. Integrated GO, KEGG, and GSEA analyses pinpointed the enrichment of differentially expressed genes in defense response pathway. Functional association network construction revealed MAPK cascade components and PR protein as hub regulators of MGB1-mediated immune signaling. MGB1 and scaffold protein ZmRACK1 together with MAPK cascade components coordinately modulated maize immune responses. We built a modulatory hierarchy of Gβ subunit at transcriptome and interacting scales, which is informative for our understanding of the regulatory basis of G protein signaling.